Hand, foot, and mouth disease (HFMD) mostly affects children under 5 years old with clinical presentation of painful blisters and lesions in th oral cavity and on the extremities. It is a major health concern in day care and school facilities due to its highly contagious nature and painful symptoms that result in dehydration and weight loss. The primary causes of HFMD are type A Enteroviruses and the emerging enterovirus 71, but there are no vaccines available for HFMD. The project described in this proposal is aimed at improving this situation by using protein engineering to alter viral polymerase fidelity and generate genetically stable attenuated viruses that can serve as candidates for live-attenuated vaccine strains. It is an interdisciplinary effort involving structural biology, polymerase biochemistry, in vitro virology to assess virus growth, and in vivo virology in a mouse model. The project is rooted in the atomic level structures of viral polymerase-RNA complexes and understanding the molecular mechanisms whereby these low fidelity viral enzymes select nucleotides and close their active sites for catalysis. These structures have already been used to engineer viral polymerases and demonstrate that either increasing or decreasing replication fidelity will attenuate virus growth in vivo, leading to the hypothesis that engineering fidelity variants can be a tool for rapidly generating safe RNA virus vaccine strains. We will demonstrate this by structure-based engineering of EV71 and Coxsackievirus A6 and A16 polymerases and testing the immunogenicity of the resulting viruses in mouse adapted viruses.